Microparticles loaded with proteins are known and are often used as a solid phase in medical, immunological, and diagnostic test procedures. The unloaded initial particles (also referred to as beads) are mainly composed of latex, e.g., polystyrene latex, and can often be magnetized due to a content of magnetite or a core made of magnetite. Proteins are usually coupled to the latex particles in a well-known manner by means of chemical linkers (covalent binding) or by adsorption (non-covalent binding).
The covalent coupling methods described in the prior art use microparticles (functionalized particles) as a starting material which have various functional groups (—COOH, -tosyl, -epoxy etc.). These functional groups are used to form covalent bonds with the proteins, e.g., via amino or carboxy groups on the proteins to be coated.
Covalent coupling methods differ from adsorptive coupling methods in that the functionalized particles used in the former case have a considerably more hydrophilic surface than non-functionalized particles. This reduces the amount of adsorptively bound protein. Adsorptively bound proteins can cause bleeding depending on the binding strength. “Bleeding” means that protein that is unbound or only weakly bound becomes detached. Only protein that has covalently reacted with the functional groups on the particle surface is permanently bound.
However, the initial particles used for covalent coating methods exhibit a high degree of lot to lot variability with regard to the density of functional groups, and the functional groups on the surface have a low storage stability which results in low loading densities and/or very variable results after loading. Another disadvantage of covalent coating methods is that the spatial accessibility of the functional groups is often poor. For this reason the loading densities required for an application, e.g., in a sensitive immunoassay, are often not achieved with particles coated in this manner. Covalent chemical coupling can also result in an inactivation of the coated protein.
Giese discloses coupling methods in U.S. Pat. No. 4,478,914 and U.S. Pat. No. 4,656,252 which enable a multilayer loading of surfaces with functional protein. In these methods biotin was covalently bound to the surface, subsequently avidin and a biotin-coupled extender was repeatedly bound to the loaded material, and unbound substance was removed by washing. In such a multilayer method a delayed bleeding can occur due to delayed desorption.
Particles with a hydrophobic surface are usually used for adsorptive coating methods. Known examples of such particles are polystyrene particles that are free from the functional groups discussed above or polystyrene particles that are derivatized with polyurethane.
Experience shows that particles that have been manufactured by adsorptive coating tend to have a higher bleeding tendency than covalently coated particles. This increased bleeding is due to the fact that the adsorptive coating occurs by means of relatively weak ionic and van der Waals interactions.
A number of tricks are known from the prior art for improving the adsorptive binding of proteins to surfaces and reducing the bleeding tendency. DE 19924643 describes the coating of particles at elevated temperatures and subsequent irradiation by UV light. These measures reduce the bleeding tendency.
Conradie, J. D. et al., J. Immunol Methods 59 (1983) 289-99 report a more efficient coating of microtitre plates by using elevated temperatures, high salt concentrations, or under acidic pH conditions.
Ishikawa, E. et al., J. Immunoassay 1 (1980) 385-98 also describe the advantageous use of strongly acidic pH conditions. They also recommend pretreating antibodies at pH 2.5 for coating polystyrene beads.
An additional disadvantage of the methods known from the prior art is the strong tendency of the hydrophobic particles to aggregate. The basic hydrophobic structure of the particles results in an increased tendency of these particles to form aggregates with one another which has a disadvantageous effect on the measurement accuracy in subsequent methods of determination using these particles. Furthermore these particles also have an increased tendency to unspecifically bind sample components when used in immunological tests due to their hydrophobic properties. Unspecific binding in such test systems is well-known to have a negative effect on test characteristics such as the signal-to-noise-ratio, or it can lead to false-positive and also to false-negative results.
In conventional wash steps the loaded particles are sedimented by centrifugation or magnetic separation and subsequently resuspended. The centrifugation or sedimentation has the effect that the loaded particles come very close to one another and in an unfavorable case form aggregates.